Eukaryotic DNA transposons can be classified into distinct superfamilies (Kapitonov & Jurka, 2004). One of the most widely distributed is the so-called hAT superfamily, which has active members in plants and insects. We began our structural studies of eukaryotic DNA transposases with Hermes, a hAT transposon that is active not only in the house fly from which it was isolated but also in other insects such as Aedes aegypti (Sarkar et al., 1997), the mosquito species that transmits yellow fever. A close relative of Hermes, the Herves transposon, is active in the malaria vector Anopheles gambiae (Arensburger et al., 2005). An active insect transposon is particularly interesting because it offers the potential to produce transgenic insects for controlling medically significant pests. Hermes transposition has been recapitulated in vitro and shown to employ a mechanism in which excision is accompanied by hairpin formation on the DNA flanking the transposon (Zhou et al., 2004), as also seen for the RAG1/2 recombinase of the adaptive immune system. In 2005, we determined the structure of an N-terminally truncated version of the 612-residue Hermes protein (Hickman et al., 2005). Hermes was shown to be a multidomain protein organized around the RNase H-like catalytic core characteristic of DDE transposases. The DDE catalytic core is disrupted by a large insertion domain whose presence conforms to the trend that DDE transposases capable of forming hairpins on their DNA substrates require an ancillary domain to provide the amino acids needed to promote hairpin formation and to stabilize them. We have recently determined the structure of a catalytically active version of Hermes bound to its transposon ends to 3.3 A resolution (Hickman et al., 2014). The resulting octameric transpososome structure allows us to propose a model for how the two differing transposon ends are bound, and we are currently testing this model. We have also solved the structure of a dimeric form of Hermes in complex with transposon ends and their adjacent flanking sequence, providing insight into how flanking DNA hairpins are formed. Other DNA transposition systems of interest to us include those that function in mammalian cells such as Tol2 from the medaka fish and piggyBac, an active moth transposon (Wu et al., 2006; Mitra et al., 2008). Another superfamily of eukaryotic DNA transposons that we have been studying are the helitrons. Although no currently active helitrons have been identified, they must once have been very active, as their remnants are widespread throughout the eukaryotic kingdom. Unlike other known eukaryotic DNA transposons, helitron insertions in the host genome are not bordered by target site duplications (TSDs), suggesting a transposition mechanism different from the common cut-and-paste mode of transposition. One particular intriguing and unique feature of Helitron transposition is its apparent ability to capture and mobilize DNA segments that may include genes located just adjacent to a Helitron. In place of the usual RNase-H superfamily type catalytic domain, helitron-encoded transposases contain a nuclease domain belonging to the HUH superfamily. We have resurrected a mammalian helitron transposon from the bat genome, and are in the process of characterizing its properties both in vitro and in vivo. Dupuy, A.J., Akagi, K., Largaespada, D.A., Copeland, N.G., and Jenkins, N.A. (2005) Nature 436, 221-226. Hickman, A.B., et al. (2005) Nat. Struct. Mol. Biol. 12, 715-721. Hickman, A.B., et al. (2014) Cell 158, 353-367. Wu, S.C., et al. (2006) Proc. Natl. Acad. Sci. USA 103, 15008-15013. Kapitonov, V.V. and Jurka, J. (2004) DNA Cell Biol. 23, 311-324. Mitra, R., Fain-Thornton, J., and Craig, N.L. (2008) EMBO J. 27, 1097-1109. Sarkar, A., Yardley, K., Atkinson, P.W., James, A.A., and O'Brochta, D.A. (1997) Insect Biochem. Mol. Biol. 27, 359-363. Zhou L.Q., et al. (2004) Nature 432, 995-1001.